The internal organization and dynamics of eukaryotic cells is largely determined by the framework of cytoskeletal elements and molecular motors that transport organelles and macromolecular complexes to specific destinations in the cell. Long range transport is generally mediated by microtubule-based motors, and short range transport and capture by the actin-based motors, most notably the myosin-V motors. The myosin- V family of molecular motors are evolutionarily highly conserved between animals, plants and fungi, and defects in vertebrate myosin-Vs cause disease. Many different organelles are transported by myosin-Vs, but how they recognize specific cargos, transport and release them at their destinations is poorly understood. To address these questions, we use budding yeast where the myosin-V encoded by Myo2 mediates transport of multiple cargos, as does its homologs Myo5A/B/C in mammals. Transport by Myo2 of two cargos is essential in yeast: transport of secretory vesicles for growth, and transport of mitochondria into the bud for segregation. Our recent work has established the Myo2 activation/inactivation delivery cycle for its major cargo of secretory vesicles, and defined redundant pathways for association of secretory vesicles and mitochondria with Myo2. In the first aim, we explore the molecular details and individual contributions of the redundant associations of Myo2 with secretory vesicles, and in the second aim we undertake a similar analysis for the association of mitochondria with Myo2. These studies will provide the most detailed view of how any molecular motor associates with its cargo. They will employ genetic and proteomic approaches integrated with quantitative live- cell imaging to define how these components contribute to organelle delivery by Myo2. For secretory vesicles to be successfully transported and then undergo exocytosis for cell growth, delivery by Myo2 has to be integrated with events during exocytosis. We have established a system to image this process at the level of a single secretory vesicle. Our third aim makes use of this ability to explore the timing and define dependencies during delivery, motor release, tethering and membrane fusion. The contributions of each component, in terms of both abundance and function, can be assessed to reveal the underlying molecular mechanisms. Our preliminary results, and anticipated findings, will provide the first in vivo analysis of how event during exocytosis at the plasma membrane are coordinated. Since essentially all the components involved are highly conserved, our findings are expected to widely applicable. Overall, this study will provide unprecedented insights into the principles of cargo recognition and delivery mechanisms by a myosin-V, which will be of broad relevance due to the high conservation between fungal and vertebrate myosin-Vs as well as their dysfunction in diseases such as Griscelli's syndrome.